Influence of Monovalent Cation Identity on Parvalbumin Divalent Ion-Binding Properties

Abstract

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na(+) and K(+) solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degrees C, the macroscopic Ca(2+)-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M(-1) and the Mg(2+) constants, 1.8 x 10(4) and 4.3 x 10(3) M(-1). In Hepes-buffered KCl, the Ca(2+) values increase to 2.9 x 10(9) and 6.6 x 10(8) M(-1) and the Mg(2+) values to 2.2 x 10(5) and 3.7 x 10(4) M(-1). Monte Carlo simulation of alpha binding data-employing site-specific constants and explicitly considering Na(+) binding-yields a K(Na) of 630 M(-1) and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na(+) ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na(+), the Ca(2+) constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M(-1), respectively; the Mg(2+) constants are 9.2 x 10(3) and 1.7 x 10(2) M(-1). In K(+), these values shift to 3.1 x 10(7) and 3.8 x 10(6) M(-1) and the latter to 1.4 x 10(4) and 2.9 x 10(2) M(-1). These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na(+) levels.

Identification and Characterization of New Protein Chemoattractants in the Frog Skin Secretome

“…Parvalbumin purification by ion exchange chromatography or 2-DE caused the loss of the chemoattractive properties. This is not surprising because in these experimental conditions, especially in the presence of salts or ampholytes, the affinity of parvalbumin for calcium and magnesium is strongly reduced (36). In contrast, it is well known that SDS does not disturb the Ca 2ϩ /Mg 2ϩ interaction with parvalbumin (37).…”

Crystal Structure of Ca2+-Free S100A2 at 1.6-Å Resolution

“…It has been reported earlier 47,48 that Na can compete with Ca 2 + for binding to EF hands, and a K d of 1.5 mM was determined for Na + binding to the CD site of rat apo-α-parvalbumin. 48 The Na + concentration of approximately 50 mM in the crystallization buffer indicates that the K d might be at least ∼ 20 mM, because both Na + ions in the N-terminal EF hands are fully occupied. In contrast to the Nterminal EF hand, no Na + binding was observed to the C-terminal EF hand.…”

Solution structure of Ca2+‐free rat β‐parvalbumin (oncomodulin)

“…The Ca 2+ -and Mg 2+ -binding constants for the EF site, 2.5 3 10 7 and 9.0 3 10 3 M À1 , approach those of the rat a isoform (1.2 3 10 8 and 1.8 3 10 4 M À1 , respectively). By contrast, the CD site exhibits sharply attenuated divalent ion affinity, with Ca 2+ -and Mg 2+ -binding constants of 1.5 3 10 6 and 1.6 3 10 2 M À1 (Hapak et al 1989;Cox et al 1990;Henzl et al 2004b). …”

“…As a consequence, the average Ca 2+ -binding constant increases from 1.2 3 10 8 M À1 in Na + solution to 1.4 3 10 9 M À1 in K + solution (DDG tot ¼À2.6 kcal/mol) (Henzl et al 2004b). …”